Tobacco companies discovered that adding ammonia to the cigarette manufacturing process helps convert bound nicotine molecules in tobacco smoke into free nicotine molecules. This process is known as "freebasing." Similar to the chemical process of freebasing cocaine, the end result is an enhanced effect of the drug on the user.
Quantitative Analysis of Ammonia in Whole Tobacco
Quantitative Analysis of Ammonia in Whole Tobacco
Objective:
To isolate and determine the amount of Ammonia in whole tobacco using the High Performance Liquid Chromatography HPLC by external standard method.
Introduction:
Ammonia is one of the many chemicals that exist in a cigarette. One method to separate ammonia is by cation-exchange chromatography using an external standard method. Ion exchange chromatography separates compounds based on net surface charge, which will enable us the identification and quantification of ammonia in cigarettes. Preparation of sample is done by lyophilizing and grinding the whole tobacco. The aim of this experiment is to determine the concentration of ammonia in cigarettes.
Objective:
To isolate and determine the amount of Ammonia in whole tobacco using the High Performance Liquid Chromatography HPLC by external standard method.
Introduction:
Ammonia is one of the many chemicals that exist in a cigarette. One method to separate ammonia is by cation-exchange chromatography using an external standard method. Ion exchange chromatography separates compounds based on net surface charge, which will enable us the identification and quantification of ammonia in cigarettes. Preparation of sample is done by lyophilizing and grinding the whole tobacco. The aim of this experiment is to determine the concentration of ammonia in cigarettes.
Materials and apparatus:
1) Lyophilizer (extract water content from cigarette)
2) Bench top grinder
3) 25,50,100 mL Volumetric flasks
4) High Performance Liquid Chromatograph
5) Solutions needed:
7.1 Ammonium Sulphate > 99 % purity.
7.2 Sulphuric Acid > 96 % purity.
7.3 Methanesulphonic Acid (MSA) – 100 %.
7.4 Type I water (as per ASTM D1193).
Procedure:
(A) Selection of method
In this experiment we will be using HPLC to detect the amount of ammonium ions found in whole tobacco. Concentration of ammonia will then be determined by absorbance measurement.
Column: cation exchange analytical column (50 mm X 4 mm)
Temperature: 30 °C
Mobile Phase / Gradient Elution Conditions (Tertiary Gradient System)
Mobile Phase: 0.003 N (3 mN) sulfonic acid solution
Solvent A: 0.003 N MSA.
Solvent B: Type I water.
Solvent C: 0.2N H2SO4.
Gradient: gradient using 0.2 N (200 mN) H2SO4 to a 0.05 N (50 mN) H2SO4 concentration
Flow: 1.5 mL/minute .
In order to adequately resolve sodium from the ammonium cation for quantitation, a 0.003 N (3 mN) sulfonic acid solution is used as the mobile phase. After the ammonium ion has eluted, a gradient using 0.2 N (200 mN) H2SO4 to a 0.05 N (50mN) H2SO4 concentration is used to remove any divalent cations and quaternary amines that may be present in the sample and may interfere with subsequent samples.
(B) Solution Preparation
For solution preparation, we will prepare the stock standards solution and extraction solution. Preparation of solution A and C will be used in Ion Chromatography.
1) Sulphuric Acid, 0.10N - Stock Standards Solution
1.1) Carefully add approximately 5 g of H2SO4 (96% w/w) to 900 mL of Type I water.
1.2) Mix and dilute to 1 L with Type I water.
2) Sulphuric Acid, 0.025N - Extraction Solution2.1) Carefully add approximately 1g of H2SO4 (96% w/w) to 900 mL of Type I water.
2.2) Mix and dilute to 1 L with Type I water.
3) Sulphuric Acid, 0.20N - Solution C (Ion Chromatography)3.1) Carefully add 10 g of H2SO4 (96% w/w) to 900 mL of Type I water.
3.2) Mix and dilute to 1 L with Type I water.
4) MSA 3mN - Solution A (Ion Chromatography)4.1) Carefully add 0.2 g of Methanesulphonic Acid (MSA) to 900 mL of Type I water.
4.2) Mix and dilute to 1 L with Type I water.
(C) Obtain a representative sample
Tobacco leaves are removed from the selected brand of cigarettes for analysis.
(D) Prepare a lab sample
1) Place approximately 3 g of whole tobacco into a pre-weighed scintillation vial.
Tobacco leaves are removed from the selected brand of cigarettes for analysis.
(D) Prepare a lab sample
1) Place approximately 3 g of whole tobacco into a pre-weighed scintillation vial.
2) Freeze-dry the whole tobacco in a lyophilizer for 48 hrs. Lyophilization (freeze drying) is the removal of water from frozen material.
3) After moisture is removed from the tobacco, grind the whole tobacco in a bench top grinder and weigh accurately 100 mg of ground tobacco into a 16 mL culture tube.
4) Add 10 mL of extraction solution and shake for 60 minutes. Allow mixture to settle for about an hour and filter the solution through a syringe filter into an 8 mL storage vial noting.
5) Using a micropipette, pipette 250 μL of the filtrate into a 2 mL auto-sampler vial and top it up with 1000 μL of extraction solution (1:5 dilution). (This will be our lab sample)
(E) Preparing Standard Solutions
(E) Preparing Standard Solutions
Ammonium stock solution
1) To prepare 25ml for each standard solution, firstly you will need to prepare your ammonium stock solution to be used in the standards. Accurately weigh 0.20 g of ammonium sulphate into a 50 mL volumetric flask
1) To prepare 25ml for each standard solution, firstly you will need to prepare your ammonium stock solution to be used in the standards. Accurately weigh 0.20 g of ammonium sulphate into a 50 mL volumetric flask
2) Dissolve in the Sulphuric Acid 0.10N of H2SO4 that you have prepared earlier on. This will be your ammonium stock solution for the standards.
3) Prepare the standards as followed in the table below:
Standard # | Volume from Ammonium stock solution (μL) | Final Volume (mL) | Concentration [μg/mL] |
1 | 0 | 25 | 0.000 |
2 | 75 | 25 | 3.2693 |
3 | 175 | 25 | 7.6283 |
4 | 250 | 25 | 10.898 |
4) The addition of amount of Ammonium stock solution will be top up to 25ml with your mobile phase (3 mN) sulfonic acid solution. (This will be your standard solution which will be measured.)
(F) Sample Analysis
1) Place the 4 Standard Solutions and the Sample Solution onto the rack in the HPLC auto-sampler. Take note of the location number on your vials on the rack.
2) Program the auto-sampler to inject 5µl portions of your standard solutions and sample solution in separate runs into the HPLC column.
(G) Measuring amount of analyte
1) Determine the peak areas of your standard solutions and sample solution.
2) Plot a Calibration graph of Peak Area (pA*s) against Concentration [μg/mL] and obtain an equation from the graph.
(H) Calculations
1) Calculate the concentration of the sample using the calibration graph to determine the amount of ammonia found in whole cigarette. Remember to multiply back the dilution factor.
2) Program the auto-sampler to inject 5µl portions of your standard solutions and sample solution in separate runs into the HPLC column.
(G) Measuring amount of analyte
1) Determine the peak areas of your standard solutions and sample solution.
2) Plot a Calibration graph of Peak Area (pA*s) against Concentration [μg/mL] and obtain an equation from the graph.
(H) Calculations
1) Calculate the concentration of the sample using the calibration graph to determine the amount of ammonia found in whole cigarette. Remember to multiply back the dilution factor.
Posted on: Tuesday, 31 July 2012 09:34 by Su-Lynn and Adam →
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